Retinal degenerative diseases and retinal trauma in humans cause progressive loss of retinal neurons and result in decreased visual function, in part because the mammalian retina does not intrinsically respond to the loss of neurons through the generation of replacement neurons. The NEI recently declared its Audacious Goal, to regenerate neurons and neural connections in the eye and visual system. This goal may be accomplished by developing strategies for activating endogenous retinal cells to manifest regenerative properties, and/or by transplanting replacement retinal neurons or their progenitors from exogenous sources. Challenges to achieving this Audacious Goal include identification of mechanisms for the activation of endogenous cells in mammals, establishment of retinal progenitors such that the missing neurons are replaced in correct numbers and ratios, and importantly for the present R21 application, integration of new neurons into retinal circuitry through the establishment of appropriate synaptic connections. The zebrafish has emerged as a critical model system for gaining knowledge of the retinal regenerative process - knowledge required for applying regenerative strategies to the mammalian retina. Our prior studies indicated that the regenerated retina of the zebrafish functionally supports basic reflexes and place preference behaviors, despite disorganization of the regenerated retinal laminae and failure of regenerated retina to re-establish normal two- dimensional patterns of retinal neurons within each lamina. These studies suggest that regenerating retinal neurons may re-wire themselves accurately even in the disrupted environment of the damaged and regenerating retina. Ultimately it will be important to understand the mechanisms through which re-wiring of retinal circuitry takes place. However, before we pursue any mechanisms it is necessary to generate a more complete picture of the re-wiring process during regeneration and the extent of successful recapitulation of native synaptic connections. In this R21 application we therefore propose a detailed study of the synaptic connections in undamaged, regenerating, and regenerated retinas of the zebrafish. We will focus on connectomes of retinal bipolar cells, thus permitting the analysis of inner and outer retinal circuitry, through the innovative use of several genetic and molecular tools available for the visualization of this retinal cell type and its synapses. Two hypotheses will be tested: 1) that retinal bipolar cell connectomes of regenerated retina recapitulate those of undamaged adult retina; and 2) that neurite reorganization is associated with accurate re- wiring during retinal regeneration.